Soybean (i.e., Glycine max L. Merr.) seeds are recognized to represent one of the most important oilseed crops presently being grown in the world. Such seeds provide an excellent source of vegetable oil as well as a source of protein that can serve as an alternative to animal meat products. For instance, tofu and soymilk derived from soybean seeds provide a major source of protein for the people of China and Southeast Asia.
While soybean oil represents an important worldwide food source, flavor and stability problems associated with its customary fatty acid composition may reduce its attractiveness in some applications. Soybean oil contains five different fatty acids as its major components. These five fatty acids are: palmitic acid (16:0) which averages about 11 percent by weight, stearic acid (18:0) which averages about 4 percent by weight, oleic acid (18:1) which averages about 20 percent by weight, linoleic acid (18:2) which averages about 57 percent by weight, and linolenic acid (18:3) which averages about 8 percent by weight of the total fatty acids. The stability problem which influences the flavor of soybean oil has been attributed to the oxidation of its fatty acids, and particularly to the oxidation of the linolenic acid (C18:3) component.
The unsaturated fatty acids in soybean oil are known to be susceptible to oxidation; and the polyunsaturated fatty acids, linoleic and linolenic, are recognized to oxidize more rapidly than the oleic acid component. The oxidized fatty acids apparently decompose to form volatile flavor-imparting compounds, to at least some degree. It is not clear why linolenic acid contributes so significantly to the flavor and stability of oils; however, based upon experiments using blends of oils having different percentages of linolenic acid, all oils containing more than about 1% linolenic acid or so appear to share this property to some extent. For more than 45 years, the flavor and stability characteristics of soybean oil have been attributed to the high linolenic acid level of the soybean oil (Dutton et al., J. Am. Oil Chem. Soc., 28:115, 1951).
To attempt to obviate the flavor and stability problems of soybean oil due to the linolenic acid content, various approaches have been proposed. Such processing of the soybean vegetable oil includes (1) minimizing the ability of the fatty acids to undergo oxidation by adding metal chelating agents or packaging in the absence of oxygen, or (2) the elimination of the endogenous linolenic acid by selective hydrogenation. These approaches have not been entirely satisfactory. Such additional processing is expensive, time consuming, commonly is found not to be completely effective, and frequently is found to generate undesirable by-products. Thus, while selective hydrogenation to reduce the linolenic acid content may improve oil stability somewhat, this also generates positional and geometric isomers of the unsaturated fatty acids that are not present in the natural endogenously formed soybean oil.
The inability to solve the stability and flavor problem adequately, together with the undesirable aspects of the necessary processing technology, reduces the end uses for soybeans to some degree, especially where such after processing of the endogenously formed oil is either unavailable or is economically inappropriate or where consumer attitudes discourage the use of hydrogenation.
Primarily because of the limitations of such after processing technology and because of the worldwide significance of soybean oil as a food source, considerable effort has been expended over many years to attempt to understand the genetic mechanism which controls the linolenic acid level in soybeans. Indeed, studies on this subject date back to at least 1949. According to Howell et al., as many as five different genes were thought to possibly control the linolenic acid level in soybeans (J. Am. Oil Chem. Soc., 26:126, 1949). Investigations into the biochemical mechanism suggest that linolenic acid (C18:3) results from successive desaturations of first oleic acid (C18:1) and second linoleic acid (C18:2). Thus, genes controlling at least two different desaturase systems may be involved. To date, the genes which can control the linolenic acid level of soybeans have not been fully identified, and the biochemical pathways that are involved have not been fully elucidated.
Even the mode of inheritance of linolenic acid production in soybeans sometimes is unclear because various studies over the years have presented conflicting results. For example, early investigation suggested that the linolenic acid content in soybeans was maternally controlled. A later study suggested the mechanism of inheritance was even more complicated, being partially maternally and partially embryonically controlled (Wilson et al., "Regulation of Linolenic Acid in Soybeans and Gene Transfer of Hiph Yielding, High Protein Germplasm", R. A. Baldwin (Ed.), Proceedings of the World Conference on Emerging Technologies in the Fats and Oils Industry, Am. Oil Chem. Soc., Champaign, Ill., (1986). The Wilson et al. study thus reports that the genes which regulate oleic acid desaturation are controlled by the maternal parent, while the genes which control linoleic acid desaturation are governed by the embryonic genotype.
Complications also have arisen because it has been long recognized that the linolenic acid content of soybeans sometimes can be influenced by the environment in which the seeds are grown (Howell et al., Agron. J., 45:526, 1953). Such environmental factors are said to include temperature, photoperiod (i.e., day length), the geographical location. and the planting date.
In summary, despite the substantial effort over the years, the genetic mechanism for controlling the linolenic acid content in soybean vegetable oils is not fully understood. Genetic research to provide soybeans characterized by reduced levels of linolenic acid is thus quite complex. There is little to guide efforts of this sort on a reliable basis. Research efforts accordingly have been largely empirical with no assurance of success.
However, despite the relative lack of understanding of the genetic mechanism which controls the level of linolenic acid content in soybeans, substantial work over the years has been carried out to attempt to isolate soybean lines having low levels of endogenously formed linolenic acid. as well as to attempt to use genetic manipulation to develop soybean lines characterized by low levels of linolenic acid. The lowest level of linolenic acid in the oil of natural soybean germplasm accessions was found by some researchers to be 4.2 percent by weight (Kleinman and Cavins, J. Am. Oil Chem. Soc., 59:305A, 1982).
Tripathi et al., Indian J. Ayric. Res., 1975, 9(4):220-222, "Note On The Quality Constituents Of Soybean (Glycine Max (L) (Merrill) Varieties", did report, among other things, the fatty acid contents of what were stated to be twelve soybean varieties grown at the Oilseed Research Farm, Kalianpur, Kanpur, during kharif, 1970. While the linolenic acid contents reported vary from 0.0 to 5.3 percent by weight, such contents were calculated by the Scholfield and Bull formulae (Tripathi et al., referencing Bailey, 1945, Industrial Oil and Fat Products, Interscience Publishers, Inc., New York). In general, Scholfield and Bull's methodology predicted fatty acid composition from the iodine value. Their data points were scattered about these linear predictions, and for linolenic acid a standard error of 1.5 percent by weight was reported. Moreover their method was standardized on lines with typical soybean oil. There is no evidence that their formula is applicable to oils having atypical compositions produced by mutation. Indeed, it would be surprising if their formula was applicable to such samples. It should be noted also that the equation of Scholfield and Bull was based on the iodine-thiocyanogen method which is recognized to be subject to considerably more error than gas liquid chromatography when measuring fatty acid composition.
In the first place, the public availability of the twelve soybean varieties referenced by Tripathi et al. is uncertain. Applicants have made repeated attempts to obtain samples of such varieties and have not been successful in securing all of them.
Secondly, and importantly, what has been determined on the basis of samples provided is that there is no correlation between the linolenic acid values reported by Tripathi et al. and those determined by gas liquid chromatography. Gas liquid chromatography is recognized to be the current and most reliable analytical standard used for the fatty acid analysis of a vegetable oil. Set forth below, for all samples obtained, is a comparison of the linolenic acid values reported in Tripathi et al. with those obtained by gas liquid chromatography (GLC).
______________________________________ Tripathi et al. GLC Values (weight percent) Values Seed From Seed From Variety (weight percent) U.S..sup.1 India.sup.2 ______________________________________ Bragg 4.5 7.6 5.8 Type 49 4.9 -- 5.9 Lee 3.7 6.7 4.0 Improved Pelican 2.4 7.2 7.0 Punjab-1 0.4 6.1 5.6 IC2716 1.0 -- 4.4 Type 33 5.3 -- -- Type 64 0.0 -- -- Type 1 1.4 -- -- IC217 2.8 -- 5.9 IC222 4.1 -- -- IC213 4.2 -- -- ______________________________________ .sup.1 Seed produced in United States and obtained from USDA, Soybean Production Research, Stoneville, Mississippi. .sup.2 Seed obtained from the National Bureau of Plant Genetic Resources, New Delhi, India.
In summary, based upon what Applicants have found, there would be no sound basis for concluding that any of the varieties referenced by Tripathi et al. possessed extremely low linolenic acid contents when such contents are determined by gas liquid chromatography. Rather, these varieties appear to have rather typical to slightly reduced linolenic acid contents. These linolenic acid contents tend to be above the minimum later reported by Kleinman et al. for natural soybean germplasm accessions.
Hybridization work to reduce the linolenic acid of soybeans dates back at least as far as 1961. White et al. identified an F.sub.2 plant obtained by hybridization with only 3.35 percent by weight linolenic acid (White, Quackenbush and Probst, "Occurrence and Inheritance of Linolenic and Linoleic Acid in Soybean Seeds", J. Am. Chem. Soc., Vol. 38, pages 113 to 117, 1961). However, this level was not maintained in succeeding generations and accordingly there is no evidence of genetic control.
During 1975 the present Applicants utilized recurrent selection to produce soybean strains having levels of linolenic acid of about 5.5 percent by weight (Fette Seifen Anstrichm., 77:97 101, 1975). Wilson and Burton isolated two different lines, designated N78-2245 and PI123440. These lines were selected for their levels of oleic acid, linoleic acid and linolenic acid. From this experimentation, two genetic systems were discovered, one that primarily governs oleic acid desaturation and a second that acts genotypically upon linoleic acid desaturation. These two gene loci are said to determine the low linolenic acid content (Crop Science, 21:788, 1981).
Wilcox et al. treated soybeans with ethyl methane sulfonate (EMS) to produce a mutant designated C1640 (J. Am. Oil Chem. Soc., 61:97, 1984). The level of linolenic acid averaged 3.4 percent by weight. It was stated that the linolenic acid trait could be transferred to other lines by backcrossing.
In our commonly assigned U.S. Pat. No. 5,534,425 soybeans are disclosed which form a reduced endogenous linolenic acid content. Also, in our commonly assigned U.S. Pat. No. 5,530,183, soybean variety 9253 is disclosed that provides a specialty oil having a reduced endogenous linolenic acid content.
F. G. Dollear et al. reported in Oil & Soap, 15:263-264, (1938) that soybean oil from the Dunfield cultivar grown at Columbia, Mo., U.S.A. in 1936 had a very low iodine value and by using the iodine and thiocyanogen values, they calculated a linolenate content of 2.9 percent. When grown at Lafayette, Ind., U.S.A. in 1937, the oil measured 6.0 percent linolenate. They attributed the low linolenate percentage to variation in growing conditions since 1936 set heat records. B. D. Rennie et al. in J. Am. Oil Chem. Soc., 66:1622 (1989) stated that high temperatures can cause depression of the linolenate content. When the Dunfield cultivar was grown in Iowa, U.S.A., it did not exhibit an abnormally low linolenate content as determined by gas liquid chromatography.
Despite these efforts there has remained a need for soybeans having a still further genetically-controlled reduction of linolenic acid in the endogenously produced vegetable oil produced within the seeds.
It is an object of the present invention to provide under conventional field growing conditions soybean seeds possessing while under genetic control a reduced level of linolenic acid in the endogenously produced vegetable oil wherein the genetic control is attributable to a new allele.
It is an object of the present invention to provide soybean plants capable upon self-pollination of forming seeds that possess while under genetic control a reduced level of linolenic acid in the endogenously produced vegetable oil wherein the genetic control is attributable to a new allele.
It is an object of the present invention to provide a vegetable oil derived from soybeans following crushing and extraction that exhibits while under genetic control (as described) a further reduced concentration of linolenic acid wherein the level of linolenic acid is less than that previously available in an endogenously formed soybean seed oil.
It is another object of the present invention to provide in soybeans a novel heretofore unknown homozygous recessive fan3fan3 gene pair that is capable of reducing linolenic acid production in the endogenously produced vegetable oil present in the seeds.
It is another object of the present invention to combine the homozygous recessive gene pairs (1) fan1fan1 or fan1(A5)fan1(A5), (2) fan2fan2, (3) fan3fan3 in a single soybean plant which in combination have been found to make possible the expression, while under genetic control, of a lesser concentration of linolenic acid in the endogenously formed vegetable oil formed in the soybean seeds of such plant than has heretofore been possible.
It is a further object of the present invention to provide a vegetable oil derived from soybean seeds that contains a reduced endogenously produced linolenic acid content while under genetic control which is particularly suited for uses requiring enhanced resistance to the development of oxidized flavors, such as products requiring a long shelf-life, or which are routinely subjected to rigorous conditions during use including cooking or frying oils.
These and other objects, as well as the scope, nature, and utilization of the claimed invention will be apparent to those skilled in the art from the following detailed description and appended claims.